N-acetylneuraminic acid (Neu5Ac) is the most common member of the sialic acid family of aminosugars. Neu5Ac is frequently found as a terminal sugar in cell surface complex carbohydrates and plays a major role in many biological processes such as cellular adhesion and binding of toxins and virus (Varki, 1993). Neu5Ac is also a major component of the carbohydrate portion of gangliosides which are notably abundant in brain tissue and are involved in several pathologies (Zhang & Kiechle, 2004).
In reason of their important biological functions, sialic acid containing oligosaccharides has attracted considerable interest and many methods have been developed to synthesize these structures for fundamental research and potential therapeutic applications. However, large scale production of sialylated oligosaccharides has not been reached as of today.
Chemical syntheses are not practical in reason of the multiple protection and deprotection steps and a lot of efforts has been put on enzymatic and biotechnological methods. Sialyltransferases use CMP-Neu5Ac as the activated sugar-nucleotide and the development of efficient processes for enzymatic syntheses of sialylooligosaccharides has been possible through the identification of bacterial sialyltransferase genes which are well expressed in E. coli and the design of multiple enzymatic systems that mimic the natural pathway of sugar nucleotide biosynthesis (Gilbert et al., 1998).
A significant improvement later came from the use of living bacterial cells to produce sialylooligosaccharides (Priem et al., 2002). In this approach, sialyllactose was directly produced by growing cells of metabolically engineered Escherichia coli strains overexpressing the Neisseria meningitidis genes for α-2,3-Sialyltransferase and for CMP-Neu5Ac synthase. The bacteria were grown at high cell density with glycerol as the carbon and energy source, while exogenous lactose and Neu5Ac were supplied as precursors for sialyllactose synthesis. During the growth, lactose and Neu5Ac were actively internalized by. E. coli β-galactoside and Neu5Ac permeases. To prevent catabolism of lactose and Neu5Ac, mutant strains devoid of β-galactosidase and Neu5Ac aldolase activities were used. Lactose and Neu5Ac accumulated in the cytoplasm where Neu5Ac was then converted into CMP-Neu5Ac to be further transferred on lactose to form sialyllactose (our European patent EP 1194584). This system was applied to the production of the carbohydrate portion of gangliosides GM2 and GM1 by additionally expressing the appropriate glycosyltransferase genes (Antoine et al., 2003). Polysialylated oligosaccharides (GD3 and GT3 sugars) were also produced by this method and with the Campylobacter cstII gene that encodes a bifunctional α-2,3- and α-2,8-sialyltransferase (our application U.S. 60/690,837 and Antoine et al., 2005).
Large scale production of sialylooligosaccharides by this microbiological method requires important amount of sialic acid as a precursor. Sialic acid can be purified from natural sources such as milk and egg yolk, but the yields are low and the procedure is not suitable for large scale production. Sialic acid is generally prepared by enzymatic synthesis by the sialic acid aldolase using N-acetylmannosamine (ManNAc) and pyruvate as substrate. To reduce the cost, ManNAc is usually prepared by chemical or enzymatic epimerization of N-acetylglucosamine which is a cheaper substrate than ManNAc (Lee et al., 2004; Maru et al., 1998). In spite of these improvements the sialic acid cost is still relatively high and this cost hampers the development of a economical system for the production of sialylooligosaccharides.
Also, strains like E. coli K1 and N. meningitidis are able to produce CMP-Neu5Ac but they are pathogenic and cannot be used in biotechnological processes for safety reasons. Most of other bacteria, including E. coli K12, do not have the enzymatic machinery for the biosynthesis of CMP-Neu5Ac, and it is a goal of the invention to genetically engineer non pathogenic strains which would be able to produce CMP-Neu5Ac from endogenous UDP-GlcNAc.
In connection with the present invention, we have designed a new microbial system for cost-effective large scale production of sialylooligosaccharides without the need of an exogenous supply of sialic acid. The metabolically engineered microorganisms of the invention are viable, non-pathogenic and can be used in large scale and industrial culture processes. They have optimized modified pathways and deletion of futile metabolic cycles and they lead to biosynthesis of activated CMP-Neu5Ac which serves as in situ sialic acid donnor to form sialylated oligosaccharides.